One may now prepare polypeptides of short and medium length by systematic, even automated, techniques. A large number of small polypeptide hormones, exhibiting potent biological activity, may be synthesized directly using automated peptide synthesizers, solid-state resin techniques, and the like. Hormones and growth factors such as epidermal growth factor, growth hormone, growth hormone releasing factor, somatostatin, vasopressin, enkephalins, endorphins, bradykinnins, and the like are all small enough to be easily accessible using current technology. Additionally, defined antigenic epitopes may be synthesized as short or medium-length polypeptides for use in vaccines. However, small polypeptides in general enjoy only a short half-life once administered, and are rapidly degraded by endogenous proteases. The therapeutic potential of such polypeptides would be dramatically increased by extension of the in vivo half-life.
In addition to increasing the half-life of peptides, other characteristics of peptides might be changed in ways which would provide useful pharmaceutically active compounds, e.g., improving binding affinity. Some of the general means contemplated for the modification of peptides are outlined in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins--A survey of Recent Developments, Weinstein, B., ed., Marcel Dekker, Inc., publ., New York (1983), which is incorporated herein by reference to disclose methods of modifying amino acids and peptides.
The substitution of D-amino acids for the normal L stereoisomer has been carried out in an attempt to increase half-life. The theory of this approach is that the proteolytic enzymes responsible for cleavage may be stereospecific, so that substituting a D-amino acid may render the peptide unacceptable as a cleavage substrate. However, the substitution may also destroy the peptide's activity, by rendering it unacceptable as a ligand for its normal receptor. R. M. Freidinger et al., Pept Chem 1987 539-48, prepared cholecystokinin cyclic peptide analogs using D-Trp. N. Sakura et al., Pept Chem 1987 557-60, prepared a series of neurokinin B analogs, using D-Ala, D-Trp, and D-Phe. J. Rivier et al., Pept Chem 1987 597-600, prepared a series of corticotropin releasing factor (CRF) peptide analogs, in which an amino acid was replaced by its D analog, and reported activities for the analogs ranging from 0.05 to 50% of the native CRF activity. Although D-isomer amino acids may be commercially available, they require either stereo-specific synthesis or a stereoresolution step for preparation. Also, since D-isomer amino acids are constrained to the opposite orientation, their use may not result in structures resembling the native peptide conformation.
Another approach to the problem has been to modify the peptide bonds that are susceptible to cleavage, for example, by replacing the amide with a saturated amine. See, for example, Skiles et al., U.S. Pat. No. 4,496,542. J. S. Kaltenbronn et al., "Proceedings of the 11th American Peptide Symposium" (ESCOM Science Publishers, The Netherlands, 1990) pp. 969-70, disclosed peptides in which all of the peptide bonds were replaced with saturated amine bonds.
Others have prepared derivatized polypeptides, typically by acetylation or alkylation. J. T. Suh et al., Eur J Med Chem--Chim Ther (1985) 20:563-70, disclosed Lys-Gly dipeptide derivatives for inhibition of angiotensin-converting enzyme, in which the Gly amide nitrogen was substituted with 2,3-dihydro-1H-indene. Sempuku et al., JP 58/150,562 (Chem Abs (1984) 100:68019b), disclosed N-substituted glycine derivatives useful for inhibition of angiotensin-converting enzyme. J. D. Young et al., "Proceedings of the 11th American Peptide Symposium" (ESCOM Science Publishers, The Netherlands, 1990) pp. 155-56, disclosed a synthesis of bradykinin, in which proline at position 7 was replaced by N-benzylglycine.
There are a number of problems with respect to using peptides as pharmaceutically active compounds. For example, the larger the peptide, the more difficult it becomes to produce commercial quantities of the peptide in sufficient purity for use as a pharmaceutically active compound. However, more importantly, with respect to the present invention, larger peptides are not particularly stable metabolically and are difficult to deliver, especially when the delivery is in convenient forms such as oral delivery systems. Further, once delivered, large peptide molecules are easily broken down by endogenous enzymes. Thus, the pharmaceutical industry still prefers to employ organic chemicals (i.e., smaller molecules which are not so difficult to synthesize and purify) rather than peptides. The number of possible organic molecules capable of interaction with any given receptor is less limited than the number of peptides meeting the same criteria. Further, organic molecules are frequently less susceptible to metabolism than are peptides, and may often be administered orally. However, it is difficult to rationally design an organic molecule for optimal activity and/or binding to a particular site.
One approach to the discovery of new pharmaceutically active organic drugs (i.e., compounds with the 3-D structure needed for binding) relies primarily on X-ray crystallography of purified receptors once the binding site is identified, organic molecules are designed to fit the available steric space and charge distribution. However, it is often difficult to obtain purified receptors, and still more difficult to crystallize the receptor so that X-ray crystallography may be applied. It is also nontrivial to devise an appropriate ligand, even after the binding site has been properly identified. Overall, it is extremely difficult to design useful pharmaceutically active compounds due to a number of factors such as the difficulty in identifying receptors, purifying and identifying the structures of compounds which bind to those receptors and thereafter synthesizing those compounds.
Another approach to the discovery of new pharmaceutically active drugs has been to synthesize a multiplicity of short peptides, followed by an assay for binding (or other) activity. R. A. Houghten, Proc Nat Acad Sci USA (1985) 82:5131-35, described a method for synthesizing a number of peptides by the Merrifield method. In the general Merrifield method, the C-terminal amino acid of the desired peptide is attached to a solid support, and the peptide chain is formed by sequentially adding amino acid residues, thus extending the chain to the N-terminus. The additions are performed sequentially by deprotecting the N-terminus, adding the next amino acid in protected form, deprotecting the new N-terminus, adding the next protected amino acid, etc. In Houghten's modification, C-terminal amino acids bound to supports were placed in individual polyethylene bags, and mixed and matched through the addition cycles, so that twenty bags (each containing a different C-terminal residue bound to a support) can be simultaneously deprotected and treated with the same protected amino acid. In this manner, one can obtain a set of peptides having different sequences simultaneously. The peptides are then recovered and tested for activity individually.
A modification of Houghten's approach was described by H. M. Geysen et al., Proc Nat Acad Sci USA (1984) 81:3998-4002 (see also WO86/06487 and WO86/00991), in which the C-terminal amino acids are supported on pins. This enabled Geysen to assay biological activity (in the form of antibody binding) without removing the peptides from the support.
Earlier methods make it possible to more quickly synthesize larger numbers of peptides than was possible previously. However, the peptide syntheses are carried out in isolated reaction conditions to produce individual peptides under each of the reaction conditions. Accordingly, these approaches do not produce mixtures of peptides containing large numbers of different peptides or peptide libraries which can be screened for the desired active peptide.
Rutter et al., PCT WO89/10931, described a method by which one can generate a large number of peptides systematically in approximately equimolar amounts, and assay the resulting library of peptides for biological activity. Rutter also disclosed using amino acids having altered side chains, such as phenylglycine, hydroxyproline, and .alpha.-aminobutyric acid. The active peptides are selected from the remainder of the library by a variety of methods, the most straightforward of which is binding to a ligand or receptor. For example, if the target peptide is to be ligand for a receptor, the library of peptides may be applied to a quantity of receptor bound to a solid support. The peptides which bind with highest affinity can then be separated from peptides with lower affinity by standard techniques. This method allows one to probe a binding site with a very large number of peptides.
Another method of producing large numbers of peptides is taught in U.S. Pat. No. 5,182,366, which application is incorporated herein to disclose methods of making large numbers of peptides. This application describes a method of preparing a mixture of peptides having a known composition and containing a peptide of a desired amino acid sequence. The method involves three essential steps. First, a given amount of a mixture of amino acyl or peptide derivatized resin is divided into a number of pools with each pool containing an equal molar amount of the resin mixture. Second, a different single amino acid is coupled to the resin mixture in each of the pools and the coupling reaction is driven to completion. The peptide mixtures in each of the pools are then mixed together to obtain a complex peptide mixture containing each peptide in retrievable and analyzable amounts. The steps can be repeated to lengthen the peptide chains and methods can be employed to retrieve the desired peptide from the mixture and carry out analyses such as the determination of the amino acid sequence. The ability to obtain mixtures with equal molar amounts of each peptide therein is dependent on the ability to accurately weigh and divide each reaction product into equal amounts and the ability to drive each reaction to completion.
Each of the above-described methods offers a method of producing one or more peptides which may have a desired biological activity. Although some of the methods may mention the use of modified amino acids, all the end products result in conventional peptide bonds. This limits applicability because only peptides are produced and if modifications are needed, substantial downstream processing is required to obtain the desired final product. Further, the peptides are subject to conventional metabolic degradation.
The present inventors postulated that there are potential nonpeptide molecules with improved protease resistance and possibly with higher affinity than the natural peptide ligand. Such nonpeptides were further postulated as less susceptible to rapid cleavage and clearance. Additionally, the present inventors postulated that such nonpeptides could be bound to pharmaceutically active organic compounds and provide conjugates which use the nonpeptide portion as biochemical targeting agents for the organic molecule bound to it.